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Journal of Bacteriology, December 2001, p. 6787-6793, Vol. 183, No. 23
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.23.6787-6793.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The Global Regulators GacA and 
S
Form Part of a Cascade That Controls Alginate Production in
Azotobacter vinelandii
Miguel
Castañeda,
Judith
Sánchez,
Soledad
Moreno,
Cinthia
Núñez, and
Guadalupe
Espín*
Departamento de Microbiología
Molecular, Instituto de Biotecnología, Universidad Nacional
Autónoma de México, Cuernavaca Morelos 62250, Mexico
Received 26 June 2001/Accepted 12 September 2001
 |
ABSTRACT |
Transcription of the Azotobacter vinelandii algD
gene, which encodes GDP-mannose dehydrogenase (the rate-limiting enzyme
of alginate synthesis), starts from three sites: p1, p2, and p3. The
sensor kinase GacS, a member of the two-component regulatory system, is
required for transcription of algD from its three sites during the stationary phase. Here we show that algD is
expressed constitutively throughout the growth cycle from the p2 and p3 sites and that transcription from p1 started at the transition between
the exponential growth phase and stationary phase. We constructed
A. vinelandii strains that carried
mutations in gacA encoding the cognate response
regulator of GacS and in rpoS coding for the
stationary-phase 
S factor. The gacA
mutation impaired alginate production and transcription of
algD from its three promoters. Transcription of
rpoS was also abolished by the gacA
mutation. The rpoS mutation impaired transcription of
algD from the p1 promoter and increased it from the p2
E promoter. The results of this study provide evidence
for the predominant role of GacA in a regulatory cascade controlling
alginate production and gene expression during the stationary phase in A. vinelandii.
| |
INTRODUCTION |
Azotobacter vinelandii is
a nitrogen-fixing soil bacterium that undergoes differentiation to form
desiccation-resistant cysts and produces two polymers of industrial
importance: alginate and poly-
-hydroxybutyrate (PHB).
A. vinelandii has been shown to posses an
alginate biosynthetic gene cluster organized in three operons (5,
25, 29, 30, 49), one of which transcribes algD, which
encodes GDP-mannose dehydrogenase, the key enzyme of the alginate
biosynthetic pathway. The algUmucABCD cluster has been
characterized in A. vinelandii and in
Pseudomonas aeruginosa and has been shown to control
alginate production (28, 34, 37, 44, 45, 53). It has been
shown for P. aeruginosa that the activity of the
alternative sigma factor E (AlgU) encoded by algU is
negatively regulated by the anti-sigma factor MucA (9, 10, 16,
27, 45, 54) and in an indirect manner by MucB (27).
In several bacterial species, E regulates
expression of functions related to the extracytoplasmic compartments
(32). In A. vinelandii,
transcription of algD can initiate at three promoters, one
of which (p2) is regulated by E (28,
34) but presumably in an indirect manner (37).
The global two-component GacS/GacA system is conserved in a variety of
gram-negative bacteria. In Erwinia carotovora and some Pseudomonas species, it controls the expression of genes
involved in secondary metabolism, phytopathogenesis, and quorum sensing (7, 8, 11, 15, 20, 24, 40, 41). In Pseudomonas syringae B728a, gacA and gacS mutations
negatively affect alginate production and algD expression
(52).
The GacS histidine kinase controls alginate production in A. vinelandii. In gacS mutants transcription of
algD is significantly reduced during exponential growth and
abolished in the stationary phase (6). Regulation of
alginate synthesis by GacS during the stationary phase was shown to be
exerted on algD transcription from its three promoters
(6).
In Escherichia coli and other bacteria, the alternative
sigma factor S (RpoS) functions as a global
regulator and is responsible for the activation of many genes expressed
mainly during the stationary phase and under various stress conditions
(18). One way in which GacA regulates gene expression in
Pseudomonas fluorescens is by influencing accumulation of
the S factor (50). In
P. aeruginosa, S
controls the production of virulence factors, such as exotoxin A,
pyocyanin, and alginate in an alginate-overproducing strain (48). A relationship between S
and quorum sensing has also been reported in P. aeruginosa (22, 51).
In E. coli transcription of rpoS in
exponentially growing cells is dependent on BarA (35).
BarA was recently identified as the cognate sensor kinase of UvrY, the
E. coli GacA homologue (39). As
GacS/GacA influences the level of S in several
bacterial species, expression of algD in A. vinelandii was proposed to be regulated by
S (6). In agreement with this
proposition, the A. vinelandii algD-p1 promoter
has the 
10 sequence CTATAAT and also has an intrinsic DNA
curvature observed in promoters preferentially recognized by
S (12, 13, 38).
Most of the two-component systems are composed of a transmembrane
histidine phosphokinase that senses environmental signals and a
cytoplasmic response regulator that activates transcription upon
phosphorylation by the sensor (19, 47).
This study reports the identification and characterization of the
A. vinelandii gacA gene, which encodes the GacS
cognate response regulator, and rpoS, which encodes the
S factor. Our data show that entering into the
stationary phase results in expression of rpoS and of
algD from its p1 promoter and that a mutation in
gacA abrogates transcription of algD and rpoS, indicating the predominant role of GacA in a
regulatory cascade that controls gene expression in the stationary
phase and alginate production in A. vinelandii.
| |
MATERIALS AND METHODS |
Microbiological procedures.
Bacterial strains and plasmids
used are listed in Table 1. Medium and
growth conditions were as follows: A. vinelandii
was grown at 30°C in Burk's nitrogen-free salts medium supplemented with 2% sucrose (21). E. coli
strain DH5
was grown on Luria-Bertani medium (31) at
37°C. Antibiotic concentrations used (in micrograms per milliliter)
for A. vinelandii and E. coli, respectively, were as follows: tetracycline, 20 and
20; kanamycin, 5 and 30; rifampin, not used and 20; ampicillin, not
used and 100; nalidixic acid, 20 and 20; spectinomycin, 100 and 100;
streptomycin, 2 and 20; and gentamicin, 1.5 and 10. A. vinelandii transformation was carried out as previously
described (3).
Alginate and PHB production was determined as previously described
(
30,
46). All measurements were done in triplicate.
Protein concentration was determined by the Lowry method
(
26).
Nucleic acid procedures.
RNA and DNA isolation and cloning,
Southern blotting, and random primer procedures were carried out as
described earlier (42). Plasmids pSAFA2 and pCNS59 were
used to determine the nucleotide sequences reported in this study. DNA
sequencing was done with the Thermosequenase sequencing kit by the
dideoxy-chain termination method of Sanger et al. (43).
Primer extension of algD and algU was carried out
as previously described (5, 37). Reactions were performed
with a primer extension system (Amersham) as instructed by the manufacturer.
Northern blot analysis.
Total RNA was extracted from the
ATCC 9046 and JM3 strains using a High-Pure RNA Isolation Kit
(Roche) and was quantified spectrophotometrically by measuring optical
density at 260 nm. For Northern analysis 10 µg of RNA was loaded per
lane. As loading and transfer controls, all blots were reprobed with a
probe specific to 16S rRNA derived from plasmid pKK3535
(4).
Cloning of A. vinelandii gacA and
rpoS genes.
Oligonucleotides gacA1
(5'-GATTAAGGTGCTGGTGGTCGACC-3') and gacA2
(5'-GCGGTGCCGTACCAGCTACGGCGG-3') and total DNA from
P. aeruginosa PAO1 were used to isolate by PCR a
fragment containing the P. aeruginosa gacA gene
(40). This fragment was used as probe to identify a cosmid
clone denoted pSMU1886, which was derived from an A. vinelandii genomic library, and contained a 3-kb
ClaI fragment that hybridized to the gacA probe.
This 3-kb ClaI fragment was cloned into the pBluescript
KS(+) vector (Stratagene) to yield plasmid pSAFA2 (Fig.
1). Oligonucleotides jsf2
(5'-TTGCCCACCTCCCGGGTGG-3') and jsf3
(5'-GCAGGGATCCAGAAAAGCCG-3') were used to isolate by PCR a
fragment containing the gacA gene. This fragment was cloned into plasmid pKT230 (2) to produce pSAFA4 (Fig. 1).
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FIG. 1.
(A) Physical map of the A.
vinelandii chromosomal
gacA-uvr region and plasmids constructed
in this study. Arrows indicate direction of transcription. Antibiotic
resistance cassette is represented by the inverted triangle. Vector
sequences are represented by black bars. (B) Physical map of
insertional inactivation of the gacA gene in
A. vinelandii ATCC 9046. Southern blot
hybridization of total genomic DNA digested with SalI
endonuclease, with the 0.8-kb SalI fragment as probe.
Lane 1, ATCC 9046; lane 2, JM3. Abbreviations: S, SalI;
C, ClaI; St, StuI.
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|
Oligonucleotides rpoS5 (5'-TTGGACGCAACGCAGCTGTATC-3') and
rpoS3 (5'-CTGGATCTGACGAACCCGCTC-3') were designed based on
the
P. aeruginosa rpoS sequence and correspond to
a
S conserved region among various species.
Total DNA from
A. vinelandii ATCC 9046 and these
oligonucleotides were used to clone by PCR
a 756-bp fragment that was
ligated into the pBluescript KS(+)
vector to yield plasmid pCNS59.
Sequence analysis of this fragment
confirmed the presence of the
A. vinelandii rpoS gene.
Construction of gacA and rpoS
mutants.
Plasmid pSAFA2 (Fig. 1), which carries a 3.0-kb
ClaI DNA fragment including gacA, was used to
construct a gacA::Gm mutation. A 0.8-kb fragment
containing a gentamicin cassette from plasmid pBSL141-Gm
(1) was inserted into the unique StuI site to
create a gacA::Gm mutation within the codon for
amino acid residue 137 of GacA. The resultant plasmid pSAFA3 (Fig. 1),
which is unable to replicate in A. vinelandii,
was introduced into strain ATCC 9046. Strain JM3, a
Gmr Aps transformant, was
selected. Plasmid PCNS59 was used to construct an rpoS
mutation. A 2-kb fragment containing a 
-spectinomycin cassette from
plasmid pHP45-Sp (14) was inserted into the unique StuI site to create the rpoS::Sp mutation within
the codon for amino acid residue 130 of RpoS. The resultant plasmid
pSMS7, which is unable to replicate in A. vinelandii, was introduced into strain ATCC 9046. Strain
CNS59, a Spr Aps
transformant, was selected and confirmed by Southern blot analysis to
carry the rpoS::Sp mutation (data not shown).
Nucleotide sequence accession number.
The nucleotide
sequences of the gacA and rpoS genes reported
here have been assigned GenBank accession numbers AF382827 and
AY029155, respectively.
| |
RESULTS AND DISCUSSION |
DNA sequence of A. vinelandii gacA
gene.
The A. vinelandii GacS sensor kinase
was previously shown to play a role as a positive regulator of polymer
synthesis, since a gacS mutation significantly reduced
alginate and PHB production. To further study regulation of alginate
production by the global two-component GacSA system, we cloned, as
described in Materials and Methods, an A. vinelandii sequence that hybridized to P. aeruginosa gacA. DNA sequence analysis of this fragment
revealed an open reading frame encoding a 214-amino-acid polypeptide
(GacA). The identity of A. vinelandii GacA was
85% with GacA present in the following Pseudomonas species:
P. syringae (41), Pseudomonas viridiflava (24), P. fluorescens (8), Pseudomonas
aureofaciens (7), and Pseudomonas tolaasii
(17). Following gacA, a partial orf
gene encoding 22 amino acids sharing similarity to UvrC, an exonuclease
that participates in DNA repair after UV damage (33), was
found. A potential Shine-Dalgarno sequence (AGGAG) is
present upstream of the gacA start codon. As in other
bacteria, the uvrC start codon overlaps the gacA
TGA termination codon (11, 33, 40), suggesting that these
two genes form an operon. As with other response regulators, GacA
contains two highly conserved aspartate residues, Asp8 and the
predicted phospho-accepting aspartate Asp54.
Alginate and PHB production is under GacA control.
As the GacS
cognate response regulator, GacA was expected to act as positive
regulator of biosynthesis of both alginate and PHB. Strain JM3, an ATCC
9046 derivative carrying a gacA::Gm mutation, was
constructed as described in Materials and Methods and was shown by
Southern blot analysis to carry the gacA::Gm
mutation (Fig. 1B). Strain JM3 was unable to produce alginate and PHB
(Table 2), confirming that GacA is an
activator of the synthesis of these polymers.
UvrC is involved in resistance to UV in both
Pseudomonas
species and
E. coli. Strain JM3 was more
sensitive to UV light than
was wild-type strain ATCC 9046 or the
gacS mutant (data not shown).
Plasmid pSAFA4 restored to the
JM3 mutant the ability to produce
alginate and PHB (Table
2) but did
not restore resistance to
UV, suggesting that the
gacA
mutation exerted polarity on
uvrC transcription and that
gacA and
uvrC are organized as an operon.
This
data also confirmed that the inability to produce alginate
and PHB is
caused by the absence of the
gacA gene product. These
results provide genetic evidence supporting the conclusion that
GacA is
the cognate response regulator of
GacS.
Growth-phase-dependent expression of algD and its
control by GacA.
In previous studies, transcription of
algD from its three promoters was documented by primer
extension experiments carried out in stationary-phase cells collected
after 48 h of growth in Burk's sucrose medium (6, 34,
36). We also reported that a gacS mutation abolished
transcription of algD during the stationary phase; however,
during exponential growth some transcription of algD
(determined by -galactosidase activity with an
algD-lacZ fusion) was detected in a
gacS mutant (6). To further study the control
of algD expression in A. vinelandii,
the transcriptional induction kinetic of algD was determined
by primer extension in cells of ATCC 9046 and the gacA
mutant JM3 throughout a growth cycle on liquid Burk's sucrose medium
(Fig. 2A). A reduction of growth was
observed in strain JM3, suggesting a GacA requirement for the control
of factors contributing to optimal growth. In the exponential phase,
algD transcription initiated from the p2 and p3 but not from
the p1 promoter. Transcription from the p1 promoter started at the
transition between exponential growth and stationary phase and
increased when cells reached the stationary phase (Fig. 2). This result
is in agreement with the hypothesis that p1 is a
S-dependent promoter.
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FIG. 2.
Growth and primer extension analysis of
algD. (A) Growth of ATCC 9046 (solid circles) and JM3
strains (empty circles) in Burk's sucrose medium. (B) Primer extension
at 8 (lane 1), 24 (lane 2), and 48 h (lane 3) incubation in
Burk's sucrose medium. (C) Hybridization of a sample of the RNA (10 µg) used as template for the primer extension with a probe specific
for 16S rRNA over 8, 24, and 48 h (4).
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|
The effect of the
gacA mutation on transcription of the
algD throughout a growth cycle is shown in Fig.
2B. Similar
to results
for the wild type, primer extension products corresponding
to
the p2 and p3 promoter but not from p1 were detected in strain
JM3
in exponentially growing cells (Fig.
2). This is an unexpected
result,
since in the
gacS mutant, transcription of
algD
(measured
as
-galactosidase activity with an
algD-
lacZ fusion) was reduced
during exponential
growth (
6). However, similar to the result
reported with
the
gacS mutant (
6), during the stationary
phase
no primer extension products corresponding to the three promoters
were detected in this
gacA mutant. This result indicates
that
the GacS/GacA system is essential for activation of the three
algD promoters during the stationary phase. These data also
imply
that control of alginate synthesis is to some extent growth phase
dependent.
algD-p1 is a S-dependent
promoter.
S is the sigma factor
responsible for the activation of many genes expressed mainly during
the stationary phase (18). As shown above, transcriptional
activation of the p1-algD promoter specifically occurs in
the stationary phase. We cloned, as described in Materials and Methods,
an A. vinelandii rpoS internal fragment encoding
amino acids 60 to 313 of S and constructed by
reverse genetics strain CNS59, a derivative of ATCC 9046 carrying an
rpoS::Sp mutation (see Materials and Methods). As predicted,
transcription of algD from the p1 promoter in the CNS59
strain was not detected (Fig. 3), confirming that p1 is a
S-dependent promoter. In addition
transcription from p2, the E-dependent
promoter during the stationary phase, was found to increase in the
rpoS mutant (Fig. 3),
suggesting that the absence of S results in
E activation. Transcription from the p3 site
was similar in the wild type and the rpoS mutant (data not
shown). The rpoS mutation did not significantly affect the
production of alginate (Table 2), suggesting that the increase in the
activity of the p1 E promoter compensates for
the negative effect on the p2 S promoter.
These data suggest that both GacA and S
participate in the same regulatory cascade and that GacA functions upstream of S.
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FIG. 3.
Primer extension analysis of algD
transcription from p1 and p2 in ATCC 9046 and CNS59 strains after 8 and
24 h of growth on Burk's sucrose medium.
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|
Growth-phase-dependent expression of rpoS and its
control by GacA.
We determined the levels of rpoS mRNA
by Northern analysis in cells of ATCC 9046 and the gacA
mutant JM3 harvested from exponential (8 h) and stationary phase (48 h)
cultures. In the wild-type strain ATCC 9046, rpoS mRNA was
detected in the stationary phase but not during exponential growth
(Fig. 4). Thus, as in other bacteria rpoS expression in A. vinelandii is
under growth phase regulation. In E. coli, for
example, the highest S concentration is found
in early stationary phase; however, a low-level expression of
rpoS as determined by Northern blot analysis is detected in
exponentially growing cells in minimal or rich media (23,
35). Correspondingly some S-dependent
genes are also expressed during exponential growth, implying a role for
S in growing cells (18). We did
not detect rpoS RNA in exponential cultures grown in Burk's
minimal medium; however, as regulation of S is unknown
in A. vinelandii, this result does not rule out a role for this factor in growing cells.
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FIG. 4.
Northern analysis of rpoS RNA isolated
from ATCC 9046 (lanes 1 and 3) and JM3 (lanes 2 and 4) after 8 and
48 h of incubation in Burk's sucrose medium.
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|
The effect of the
gacA mutation on transcription of the
rpoS is also shown in Fig.
4. No RNA corresponding to
rpoS was detected
in the
gacA mutant. Together,
these results indicate that GacA
mediates signal transduction between
GacS and the activation of
the
rpoS promoter; in turn,
S mediates activation of the
algDp1
promoter by GacA (Fig.
5).
Whether GacA
directly interacts with the
rpoS promoter region
remains to
be determined.
The gacA mutation has no effect on
algU transcription.
The lack of transcription from
p2 in JM3 during the stationary phase suggested that transcription of
algU, the gene encoding E, might be
under GacA control. We carried out primer extension analysis of
algU, with RNA isolated from strains ATCC 9046 and JM3 (data
not shown). We found that the gacA mutation has no effect on
transcription of algU; thus, stationary-phase induction of the algD-p2 promoter by GacA seems to be exerted via a
E-independent intermediary (Fig. 5).
The results of this study show that the
gacA gene cloned
encodes the cognate response regulator of GacS which is required
for
polymer synthesis and which is specifically required to activate
transcription of
algD from its three promoters, one of which
was
shown to be a
S-dependent
promoter.
Activation of gene expression by the GacS/GacA system appears to use
different signal pathways or cascades, one of which includes
rpoS, since we showed that GacA is required for
transcription
of
rpoS. By regulating expression of
rpoS, the GacS/GacA system
must play an important role in
the control of stationary-phase
functions. GacA was also shown to be
required to activate the
algD non-
S
promoters; thus, activation of alginate synthesis by GacS/GacA
is also
mediated by another as-yet-unidentified
pathway.
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ACKNOWLEDGMENTS |
This work was supported by grant 27767 from CONACyT.
We acknowledge Rene Hernandez and Josefina Guzman for technical support
and G. Soberón-Chávez for reviewing the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Microbiología Molecular, Instituto de Biotecnología,
Universidad Nacional Autónoma de México, Apdo. Postal
510-3, Cuernavaca Morelos 62250, Mexico. Phone: 52-73-291644. Fax:
52-73-172388. E-mail: espin{at}ibt.unam.mx.
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Journal of Bacteriology, December 2001, p. 6787-6793, Vol. 183, No. 23
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.23.6787-6793.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
